Method for the treatment of malignancies

ABSTRACT

A method of treating cancerous tumors is presented herein. The method includes injecting an effective dose of a plasmid encoded for IL-12, B7-1 or IL-15 into a cancerous tumor and subsequently administering at least one high voltage, short duration pulse to the tumor. The electroporation pulses may be administered at at least 700V/cm for a duration of less than 1 millisecond. The intratumor treatments with electroporation may be administered in at least a two-treatment protocol with the time between treatments being about 7 days. The intratumor treatments with electroporation may be administered in a three-treatment protocol with a time of four days between the first and second treatments and a time of three days between the second and third treatments. It was found that the intratumor treatments using electroporation not only resulted in tumor regression but also induced an immune memory response which prevented the formation of new tumors.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of and claims the benefit of U.S.application Ser. No. 13/969,078, issuing on Jan. 6, 2015 as U.S. Pat.No. 8,927,518, entitled “Method for the Treatment of Malignancies”,filed Aug. 16, 2013, which is a divisional of and claims the benefit ofU.S. Pat. No. 8,802,643, entitled “Method for the Treatment ofMalignancies”, filed on Aug. 24, 2011, which is a continuation in partof and claims the benefit of U.S. Pat. No. 8,026,223, entitled “Methodfor the Treatment of Malignancies”, filed Nov. 30, 2005, which claimsthe benefit of International Patent Application No. PCT/US2004/017153,entitled “Method for the Treatment of Malignancies”, filed Jun. 1, 2004,which claims the benefit of U.S. Provisional Patent Application No.60/320,239, entitled “IL-12 Plasmid Delivery by In Vivo Electroporationfor the Successful Treatment of Established Subcutaneous B16.F10Melanoma”, filed May 30, 2003, the contents of each of which are herebyincorporated by reference

GOVERNMENTAL SUPPORT

This invention was made with government support under Grant Number R01CA122518 awarded by National Institutes of Health. The government hascertain rights in the invention.

BACKGROUND OF INVENTION

The effective treatment of metastases is a challenge for any cancertreatment. For immunotherapy to be beneficial in the treatment ofmetastatic disease, the immune system must recognize tumor cellsthroughout the body, which can be achieved by inducing a systemic immuneresponse or through the creation of memory T cells following recognitionof a primary tumor.

Many cytokines have been intensively investigated as potentialanticancer agents. Among the many cytokines evaluated, Interleukin-12(IL-12) has been shown to exhibit strong antitumor activities. IL-12 canupregulate the proliferation and maturation of T cells and naturalkiller (NK) cells, induce production of IFN-γ, inhibit angiogenesis, andupregulate expression of accessory molecules such as HLA. Unfortunately,delivery of IL-12 in the form of recombinant protein results in severetoxicity and adverse side effects, including death. Therefore, genetherapy strategies for delivery of IL-12 have been explored such as theuse of viral vectors, gene gun, microspheres, direct injection ofplasmid, and electroporation.

The antitumor potential of IL-12 has been reported in numerousimmunotherapy studies. The proposed antitumor mechanisms of IL-12include effects on the immune system such as the induction of IFN-γ,upregulation of T cells, and proliferation of natural killer (NK) cells.In addition, IL-12 inhibits angiogenesis, the formation of new bloodvessels. This wide range of effects on the immune system as well asantiangiogenic properties results in a potentially potent antitumortreatment. Unfortunately, preclinical and clinical trials using systemicadministration of recombinant IL-12 demonstrated potential adverse sideeffects. Administration of recombinant I1-12 locally or systemically hasbeen reported to induce potent antitumor activity in a variety of murinetumor models, causing regression of established tumors. However, inthese studies, repeated delivery of recombinant IL-12 on a daily basiswas required to achieve the maximal therapeutic activity, and was alsousually associated with a dose-dependent toxicity. The use of genetherapy for the delivery of IL-12, by gene gun, resulted in fewer sideeffects than recombinant protein therapy. Several studies using viraland nonviral gene delivery techniques have reported success in slowingand/or preventing tumor growth. However, these studies have had limitedsuccess in demonstrating complete regression of the poorly immunogenicB16.F10 melanoma and subsequent resistance to challenge.

In vivo electroporation is a gene delivery technique that has been usedsuccessfully for efficient delivery of plasmid DNA to many differenttissues. Studies have reported the administration of in vivoelectroporation for delivery of plasmid DNA to B16 melanomas and othertumor tissues. Although systemic administration of recombinant IL-12revealed its antitumor potential, expression of IFN-gamma at the tumorsite has been shown to be critical for successful tumor regression.Systemic and local expression of a gene or cDNA encoded by a plasmid canbe obtained with administration of in vivo electroporation. Use of invivo electroporation enhances plasmid DNA uptake in tumor tissue,resulting in expression within the tumor, and delivers plasmids tomuscle tissue, resulting in systemic cytokine expression.

It has been shown that electroporation can be used to transfect cells invivo with plasmid DNA. Recent studies have shown that electroporation iscapable of enhancing delivery of plasmid DNA as an antitumor agent.Electroporation has been administered for treatment of hepatocellularcarcinomas, adenocarcinoma, breast tumors, squamous cell carcinoma andB16.F10 melanoma in rodent models. The B16.F10 murine melanoma model hasbeen used extensively for testing potential immunotherapy protocols forthe delivery of IL-12 and other cytokines either as recombinant proteinor by gene therapy.

Its wide range of effects on the immune system and its antiangiogenicproperties make IL-12 an excellent candidate for use an asimmunotherapeutic agent. Because of its potential toxicity, it isimportant to give careful consideration to the delivery method of IL-12.In vivo electroporation is a safe, nontoxic delivery system and has beenused for efficient delivery of chemotherapeutic agents and plasmid DNA,including plasmids encoding IL-12.

Electroporation mediated in vivo delivery of the murine interleukin-12(IL-12) gene in an expression plasmid has been shown to provideantitumor and antimetastasis activity. Various protocols are known inthe art for the delivery of plasmid encoding 11-12 utilizing in vivoelectroporation for the treatment of cancer. The protocols known in theart describe in vivo electroporation mediated cytokine based genetherapy, both intratumor and intramuscular, utilizing low-voltage andlong-pulse currents. Prior art methods have identified these low-voltagelevels to be less than 300V and long pulses to be in the area of 50 ms.Rationalization for the use of low-voltage levels and long pulse lengthsfor the delivery of plasmid encoding IL-12 for the treatment of tumorsis based on well-known principles of electroporation andelectrochemotherapy. It is known that electric pulses with moderateelectric field intensity can cause temporary cell membranepermeabilization, which may then lead to rapid genetic transformationand manipulation in a wide variety of cells types including bacteria,yeasts, animal and human cells, and so forth. Conversely, electricpulses with high electric field intensity can cause permanent cellmembrane breakdown and tissue damage. All prior art methods describingthe administration of an electroporation protocol for delivery of IL-12to the target tissue are based on the application of low-voltage, longlength pulses. These treatment protocols known in the art have not beeneffective in demonstrating acceptable cure rates for tumors, includingB16.F10 melanoma tumors. Additionally, the known treatment protocolshave been unable to demonstrate improved long-term subject survivalrates.

Accordingly, what is needed in the art is an electroporation protocolfor the delivery of a plasmid encoding a therapeutic protein that willprovide substantially improved results in the regression of cancertumors while also substantially improving the long-term survival rates.

SUMMARY OF INVENTION

The present invention provides a method for the treatment ofmalignancies, wherein the administration of a plasmid encoding for atherapeutic protein in combination with electroporation has atherapeutic effect on primary tumors as well as distant tumors andmetastases.

According to one embodiment of the invention, a method of treating asubject having a cancerous tumor is provided, the method includesinjecting the cancerous tumor with an effective dose of plasmid codingfor a therapeutic protein and administering electroporation therapy tothe tumor. The electroporation therapy further includes theadministration of at least one high voltage pulse having a shortduration.

The method of the present invention is effective in the treatment of avariety of cancerous tumors, including melanoma. The data presented isan exemplary embodiment of the present invention for the treatment ofB16.F10 melanoma in mice. However, the exemplary embodiment and datapresented are not intended to limit the method of the present inventionto the treatment of B16.F10 melanoma. The method of the presentinvention is applicable to the treatment of a variety of cancers,including, but not limited to melanoma, Merkel cell carcinoma, T-celllymphoma, squamous cell carcinoma, pancreatic cancer, and hepatocellularcarcinoma.

A variety of cytokines have been identified as being effective in thetreatment of cancer. Interleukin 12 (IL-12) is a cytokine that has beenstudied extensively as an antitumor agent. In a particular embodiment ofthe present invention, the plasmid coding for a therapeutic proteinadministered to a subject is a plasmid coding for IL-12.

In an additional embodiment, the plasmid coding for a therapeuticprotein administered to a subject is a plasmid coding for B7-1.

In an additional embodiment, the plasmid coding for a therapeuticprotein administered to a subject is a plasmid coding for IL-15.

Other effective cytokines are within the scope of the present invention.

The electroporation therapy administered in accordance with the presentinvention is characterized by high voltages pulses of short duration. Inaccordance with the present invention, a high voltage pulse is definedto be greater than about 400V/cm. Additionally, in accordance with thepresent invention a short duration pulse is defined to be less thanabout 1 millisecond.

In a particular embodiment, the electroporation therapy administered tothe subject tumor includes at least one high voltage pulse of about1500V/cm having a duration of about 100 microseconds.

In an additional embodiment, the method of the present invention furtherincludes the step of injecting an effective dose of plasmid encoding fora therapeutic protein into the muscle tissue of the subject andadministering electroporation to the subject intramuscularly using atleast one low voltage pulse having a long pulse width. The plasmidencoding for a therapeutic protein used in this step may be a plasmidencoding for IL-12, or any other effective plasmid.

In a particular embodiment of the intramuscular electroporation therapystep, the voltage level is a voltage of about 100V/cm and the pulseduration is about 20 milliseconds.

An increase in the effectiveness of the treatment has been observed whenthe treatment method of the present invention is administered multipletimes. In this instance, a method of treating a subject having acancerous tumor, is provided which includes injecting the canceroustumor with a first effective dose of plasmid coding for a therapeuticprotein, administering a first electroporation therapy to the tumor, thefirst electroporation therapy further comprising the administration ofat least one high voltage pulse having a short duration, thensubsequently injecting the cancerous tumor with a second effective doseof plasmid coding for a therapeutic protein, and administering a secondelectroporation therapy to the tumor, the second electroporation therapyfurther comprising the administration of at least one high voltage pulsehaving a short duration. Additionally, a third effective dose of plasmidcoding for a therapeutic protein and a third electroporation therapy maybe administered to the tumor, the third electroporation therapy furthercomprising the administration of at least one high voltage pulse havinga short duration. This two or three step process may be followed by thestep of injecting an effective dose of plasmid encoding for atherapeutic protein into the muscle tissue of the subject andadministering electroporation to the subject intramuscularly using atleast one low voltage pulse having a long pulse width.

A plurality of high voltage, short pulse duration electroporationtherapy conditions are within the scope of the present invention. In anexemplary embodiment, the method of the present invention includesinjecting a cancerous tumor with a first effective dose of plasmidcoding for IL-12, administering a first electroporation therapy to thetumor, the first electroporation therapy further comprising theadministration of six pulses delivered at 1500V/cm at 100 microsecondspulse duration, injecting the cancerous tumor with a second effectivedose of plasmid coding for IL-12, administering a second electroporationtherapy to the tumor, the second electroporation therapy furthercomprising the administration of six pulses delivered at 1500V/cm at 100microseconds pulse duration, injecting the cancerous tumor with a thirdeffective dose of plasmid coding IL-12, and administering a thirdelectroporation therapy to the tumor, the third electroporation therapyfurther comprising the administration of six pulses delivered at1500V/cm at 100 microseconds pulse duration. Additionally, the methodmay include injecting an effective dose of plasmid encoding for atherapeutic protein into the muscle tissue of the subject, administeringelectroporation to the subject intramuscularly using 12 pulses deliveredat 100V/cm of 20 milliseconds in duration.

In an exemplary embodiment of the present invention, a method for thetreatment of malignancies is provided wherein the method includesadministering a first treatment on day zero, the first treatmentcomprising injecting the cancerous tumor with a first effective dose ofplasmid coding for IL-12 and administering a first electroporationtherapy to the tumor, the first electroporation therapy furthercomprising the administration of six pulses delivered at 1500V/cm at 100microseconds pulse duration. On day four a second treatment isadministered comprising injecting the cancerous tumor with a secondeffective dose of plasmid coding for IL-12 and administering a secondelectroporation therapy to the tumor, the second electroporation therapyfurther comprising the administration of six pulses delivered at1500V/cm at 100 microseconds pulse duration. On day seven a thirdtreatment is administered, the third treatment comprising injecting thecancerous tumor with a third effective dose of plasmid coding IL-12 andadministering a third electroporation therapy to the tumor, the thirdelectroporation therapy further comprising the administration of sixpulses delivered at 1500V/cm at 100 microseconds pulse duration. Anadditional step may include injecting an effective dose of plasmidencoding for IL-12 into the muscle tissue of the subject, administeringelectroporation therapy to the subject intramuscularly using twelvepulses delivered at 100V/cm at 20 millisecond duration.

In an additional embodiment, delivery of plasmid encoding B7-1 tomelanoma tumors utilizes 1300 V/cm pulses of 100 μs in duration.

In an additional embodiment, Interleukin-15 (IL-15) is deliveredintratumorally utilizing electroporation. IL-15 is a 15 kDa cytokineprotein that uses the gamma and beta chains of the IL-2 receptor complexwith a unique alpha chain to signal T cells. It stimulates memory CD8+cells in contrast to IL-2, which inhibits memory CD8+ T-cellproliferation. In addition, IL-15 also inhibits IL-2-mediatedactivation-induced cell death (AICD) associated with self-tolerance.Likewise, in addition to stimulating memory CD8+ T cells, IL-15 alsostimulates the activation, proliferation and cytotoxicity of naturalkiller (NK) cells. As such, IL-15 has been targeted as an antitumorcytokine with potential advantages over IL-2.

In the present invention, IL-15 was delivered as a DNA plasmid throughelectroporation to mediate antitumor activity. In a particularembodiment, the cancerous tumor was injected with an effective dose ofplasmid coding for IL-15 and electroporation therapy was delivered tothe tumor, the electroporation therapy comprising the administration ofat least one pulse having a field strength of at least 700V/cm and aduration of less than 1 millisecond.

As demonstrated by the results provided in the detailed description, themethod of the present invention provides a treatment protocol for cancerresulting in a statistically significant improvement in survival ratesover all other known methods in the art that utilize a plasmid codingfor IL-12, IL-15 or B7-1 and electroporation. The protocol of thepresent invention utilizes high voltage, short duration pulses. Allother protocols known in the art for the delivery and expression ofIL-12, IL-15 or B7-1, utilize low voltage, long duration electroporationpulses. As such, the present invention results in new and unexpectedresults based on a novel protocol for the delivery of a plasmid codingfor a protein and electroporation.

BRIEF DESCRIPTION OF THE DRAWINGS

For a fuller understanding of the nature and objects of the invention,reference should be made to the following detailed description, taken inconnection with the accompanying drawings, in which:

FIG. 1A-B is a graphical illustration of the administration of plasmidDNA encoding IL-12 followed by electroporation results in complete tumorregression. (A) Fold increase over day 0 tumor volume followingtreatment. P, pIRES IL-12; V, control plasmid, pND2Lux; E,electroporation. Treatment mode of delivery: i.t., intratumor; i.m.,intramuscular. A plus sign indicates treatment was administered; a minussign indicates treatment was not administered. Initial treatment day isday 0; mice were treated again on day 7. Results for all groups (exceptP−E+ i.t. and V+E+ i.t.) represent the combined data from threereplicate experiments, and error bars represent the standard error ofthe mean. The P−E+ i.t. and V+E+ i.t. treatment groups were tested inone experiment because existing data in our lab showed these treatmentsto be ineffectual. Error bars for these two groups represent standarddeviation. The total number of samples for each treatment group are asfollows: P−E−, n=16; P−E+ i.t. and V+E+ i.t., n=8; and for the remainderof groups, n=17. Mice were killed when tumor volume exceeded 1000 mm3.Data are expressed for surviving mice on each day. (B) Percentagesurvival of mice represented in (A). Mice either succumbed to disease orwere killed when tumor volume exceeded 1000 mm3.

FIG. 2A-B is a graphical illustration of the results of the analysis ofserum and tumor tissue for IL-12 and IFN-γ expression. P, pIRES IL-12;E, electroporation. Mode of delivery: i.t., intratumor; i.m.,intramuscular. (A) Serum levels of IL-12 and IFN-_ in tumor-bearingmice. For each treatment group on each day tested, n=4 mice. Error barsrepresent standard deviation. (B) Mean tumor expression of IL-12 andIFN-_. For each treatment group on each day tested, n=4 mice. Error barsrepresent standard deviation.

FIG. 3A-C is an illustration of representative sections of tumor tissue,5 days after treatment, analyzed by H&E staining for infiltrating immunecells. Three sections per tumor were examined. All sections are shown at×250 magnification. An area containing immune cells is marked by a box.(A) No treatment. (B) Administration of IL-12 i.m. with electroporation.(C) Administration of IL-12 i.t. with electroporation.

FIG. 4A-F is an illustration of representative sections of tumor tissue,5 days after treatment, analyzed by immunohistochemistry for the stainedbrown. An arrow in (B) points to a cell representative of positivestaining. (A, B) Staining for CD4+ lymphocytes and CD8+ lymphocytes,respectively, from untreated tumors. (C, D) Staining for CD4+lymphocytes and CD8+ lymphocytes, respectively, from tumors receivingi.t. injection of plasmid DNA encoding IL-12 followed byelectroporation. (E, F) Staining for CD4+ lymphocytes and CD8+lymphocytes, respectively, from tumors following i.m. administration ofplasmid DNA encoding IL-12 with electroporation.

FIG. 5A-B is a graphical illustration of the administration of IL-12followed by electroporation which does not result in tumor regression ina nude mouse model. (A) Fold increase over day 0 tumor volume followingtreatment. P, pIRES IL-12; V, control plasmid, pND2Lux; E,electroporation. Mode of delivery: i.t., intratumor. Initial treatmentday is day 0; mice were treated again on day 7. The data represent twoexperiments each, with four mice in each group. Error bars representstandard deviation. Mice were killed when tumor volume exceeded 1000mm3. Data are expressed for surviving mice on each day. (B) Percentagesurvival of mice represented in (A). Mice either succumbed to disease orwere killed when tumor volume exceeded 1000 mm3.

FIG. 6A-D is an illustration of the immunohistochemical analysis oftumor tissue for the presence of blood vessels. Representative sectionsrich in vessels are depicted for each treatment. Three sections pertumor were examined. All sections are shown at _400 magnification. Anarrow in (A) points to a representative blood vessel. (A) Presence ofblood vessels within tumors on day 0, before treatment. (B) Untreatedtumors on day 5. (C) Tumors on day 5 from mice receiving i.m. injectionof plasmid DNA encoding IL-12 followed by electroporation. (D) Bloodvessels on day 5 from mice receiving i.t. administration of plasmid DNAencoding IL-12 followed by electroporation.

FIG. 7 is a table illustrating the tumor blood vessel counts fromC57BL/6 mice in each treatment group.

FIG. 8A-B is a graphical illustration of the three treatment protocol inaccordance with the present invention. For the three treatment protocol,day 0 is the day of the initial treatment and mice were treated again ondays 4 and 7. (A) Fold increase of tumor volume compared to tumor volumeon day of first treatment. (B) Percent survival of nice followingtreatment. Results represent the combined date of three replicateexperiments and error bars represent the standard error of the mean. Thetotal number of samples for each treatment group was 50. Mice wereeuthanized when tumor volume exceeded 1000 mm³. For both (A) and (B),data is expressed for surviving mice on each day. P=pIRES IL-12;V=control plasmid, pND2Lux; E=electroporation. For location oftreatment, i.t.=intratumor delivery; i.m.=intramuscular delivery.

FIG. 9A-B is a graphical illustration of the short-term prevention ofsecond tumors in accordance with the present invention. Three treatmentswere administered on days 0, 4, and 7 and two treatments administered ondays 0 and 7. (A) Percent of mice that had a tumor form on the rightflank, which received no treatment. (B) Percent survival of micefollowing treatment; 5×10⁵ B16.F10 cells were injected on the rightflank on day 0, at a time that the established tumor on the left flankwas treated. Mice were euthanized when tumor volume exceeded 1000 mm³.Data represents three replicate experiments with an n of 5 each. P=pIRESIL-12; V=control plasmid, pND2Lux; E=electroporation. For location oftreatment, i.t.=intratumor delivery; i.m.=intramuscular delivery.

FIG. 10A-B is a graphical illustration of the prevention of second tumorinduced prior to initiation of therapy. Three treatments wereadministered on days 0, 4, and 7 and two treatments administered on days0 and 7. (A) Percent of mice that had a tumor form on the right flank,which received no treatment. (B) Percent survival of mice followingtreatment; 5×10⁵ B16.F10 cells were injected on the right flank. Threedays after cells were injected on the left flank. Mice were euthanizedwhen tumor volume exceeded 1000 mm³. Data represents three replicateexperiments with an n of 5 each. P=pIRES IL-12; V=control plasmid,pND2Lux; E=electroporation. For location of treatment, i.t.=intratumordelivery; i.m.=intramuscular delivery.

FIG. 11 is a table illustrating the results of a treatment withintramuscular administration of IL-12 by electroporation and how thetreatment prevents development of tumor nodules in the lungs.

FIG. 12 is a graphical illustration of the survival rate of micereceiving a high dose of B16 cells intravenously. Three treatments wereadministered on days 0, 4, and 7. Mice were followed for 21 days andthen euthanized. Data represents two replicate experiments with an n of4 in each. Mice received an injection of 5×10⁵ B16.F10 cells to the tailvein on day 0, at the time of treatment by delivering plasmidintramuscularly. P=pIRES IL-12; V=control plasmid, pND2Lux;E=electroporation.

FIGS. 13 (A) and (B) are graphical illustrations of the results of thetreatment of melanoma tumors in a mouse model using B7-1 administeredwith an electroporation protocol of 1300 V/cm and 100 μs pulses.

FIG. 14 is a graphical illustration of the measurement of expression ofIL-15 in B16 melanoma tumor lysates after intratumoral delivery ofpIL-15 with or without electroporation. The mean concentrations oftumoral IL-15 are expressed as pg/mg tumor.

FIG. 15 is a table illustrating the initial effects of pIL-15 pluselectroporation on tumor growth.

FIG. 16 is a graphical illustration of Kaplan-Meier survival curves forC57BL/6 mice in treatment groups injected with pIL-15 or control plasmidwith or without electroporation.

DETAILED DESCRIPTION

In the following detailed description of the preferred embodiments,reference is made to the accompanying drawings, which form a parthereof, and within which are shown by way of illustration specificembodiments by which the invention may be practiced. It is to beunderstood that other embodiments may be utilized and structural changesmay be made without departing from the scope of the invention.

Materials and Methods for IL-12

Tumor Cells and Mice.

B16.F10 murine melanoma cells (CRL 6475; American Type CultureCollection, Rockville, Md.) were maintained in Dulbecco's minimalEagle's medium (DMEM) supplemented with 10% FCS and 0.2% gentamicin.Cells were trypsinized and washed in sterile PBS before injection. Theleft flank of C57BL/6 mice (National Cancer Institute, Bethesda, Md.)was shaved and 1×10⁶ cells in 50 μl of sterile PBS were injectedsubcutaneously. When challenged, mice were injected with 5×10⁵ B16.F10cells in the right flank. Tumors were measured using digital calipers,and treatment was begun when tumors reached 3-5 mm in diameter, ˜7-10days after injection. Tumor volume (v) was calculated using the formulav=a²bλ/6, where a=the smallest diameter and b=the perpendiculardiameter. Mice were housed in accordance with AALAM guidelines.

Plasmid DNA.

pIRES IL-12 was a gift from Karin Moelling (University of Zurich,Zurich, Switzerland). Briefly, pIRES IL-12 contains both subunits joinedby an internal ribosomal entry site (IRES) behind a singlecytomegalovirus (CMV) promoter. Robert Malone (Gene Delivery Alliance,Inc., Rockville, Md.) donated the pND2Lux, which encodes the reportergene luciferase. Qiagen Mega Kits (Qiagen, Valencia, Calif.) were usedfor plasmid preparations. pIRES IL-12 was prepared with anendotoxin-free kit. All plasmid DNA was diluted in sterile injectablesaline (0.9%) and stored at −20° C.

Intratumor Treatment.

Mice were anesthetized using 97% oxygen and 3% isoflurane. Tumors wereinjected with 50 μl (1 μg/ml) plasmid DNA in sterile saline using atuberculin syringe with a 25-gauge needle. An applicator containing sixpenetrating electrodes ˜1 cm in diameter was inserted into the tumor.Six pulses were delivered at 1500 V/cm (99 μs, 1 Hz) using a BTX T820pulse generator (BTX, San Diego, Calif.).

Intramuscular Treatment.

Mice were anesthetized as described earlier. The skin surrounding thegastrocnemius muscle was shaved. Plasmid DNA diluted in sterile saline(50 μl, 1 μg/ml) was injected into the gastrocnemius muscle using atuberculin syringe and a 25-gauge needle. An applicator speciallydesigned for the mouse gastrocnemius containing four penetratingelectrodes in a rectangular pattern was inserted into the musclesurrounding the injection site. A total of 12 pulses were deliveredsegmentally at 100 V/cm (20 ms, 1 Hz) using a BTX T820 pulse generator.

ELISA.

Mice were humanely killed using CO2 asphyxiation, and then blood andtumors were collected on each day from four mice per treatment group.For detection of cytokines in the serum, blood was collected by cardiacpuncture and stored at 4° C. overnight. Serum was extracted from bloodsamples by centrifugation (3 minutes at 5000 rpm) at 4° C., and storedat −20° C. until analyzed. To measure cytokine levels within the tumortissue, the tumors were removed, frozen immediately on dry ice, weighed,and then stored at −80° C. For analysis, the tumors were thawed, and 1ml of a solution containing PBS and 10% protease inhibitor cocktail(P8340; Sigma, St. Louis, Mo.) was added. The tissues were kept on ice,homogenized using a PowerGen 700 (Fisher Scientific, Pittsburgh, Pa.),centrifuged for 3 minutes at 5000 rpm at 4° C., and then supernatantswere assayed by ELISA. Both serum and tumor samples were analyzed usingmurine IFN-γ and IL-12 p70 ELISA kits (R&D Systems, Minneapolis, Minn.).Serum levels were calculated as pg of cytokine per ml of serum. Cytokinelevels in the tumor were calculated as pg of cytokine per mg of tumor.

Histology.

Mice were humanely killed by CO2 asphyxiation. Tumors were excised andplaced in 50-ml conical tubes containing 10 ml of 10% formalin. Thetissue was stained with H&E after fixation, as follows: after fixationin 10% neutral buffered formalin for 6 hours, representative tissuesamples were processed into paraffin blocks using a Miles VIP tissueprocessor (Miles Inc., Mishawaka, Ind.). Briefly, tissues weredehydrated in ascending grades of ethanol, cleared in xylene, andinfiltrated in paraffin (Tissue Prep 2; Fisher Scientific). Followingembedding, tissues were sectioned on a standard rotatory microtome and4-μm sections were retrieved from a water bath and mounted on glassslides. Three sections per tumor were examined. Sections were heat-driedand stained with H&E (Richard-Allan Scientific, Kalamazoo, Mich.) usingstandard histologic techniques. Using a synthetic mounting medium,coverslips were then placed.

Immunohistochemistry.

Immunohistochemical staining was conducted to examine the tumors for thepresence of CD4+ lymphocytes, CD8+ lymphocytes, and blood vessels usingthe following antibodies: rat anti-mouse CD4, rat anti-mouse CD8a (Ly2),and rat anti-mouse CD31 (PECAM-1), respectively (PharMingen, Cambridge,Mass.). Mice were humanely killed by CO2 asphyxiation. Tumors wereexcised with scissors and the skin removed, then immediately frozen in amixture of dry ice and ethanol, and stored at (80° C. Frozen sections of5 μm were obtained. For immunohistochemical analysis, rat antimouse CD4,rat anti-mouse CD8a (Ly2), or rat anti-mouse CD31 (PECAM-1) was appliedto tissue sections at a dilution of 1:50 and incubated for 30 minutes,followed by detection with the Vector Elite Rat IgG-Peroxidase kit at 2×concentration (15 minutes each in biotinylated anti-rat IgG and ABCcomplex). Immunostaining was carried out on the Dako autostainer.Sections were analyzed at ×400 magnification.

Treatment of Nude Mice.

BALB/c athymic nude mice were obtained from the National CancerInstitute and used at 7 weeks of age. B16.F10 cells were prepared asdescribed earlier. Mice were injected subcutaneously in the left flankwith 1×10⁶ B16.F10 cells in 50 ml of sterile PBS. Treatment was begunwhen the tumors reached 3-5 mm in diameter. Mice received intratumortherapy as described earlier.

Statistical Methods.

Statistical analysis was performed by ANOVA or two-tailed Student'st-test.

FIGS. 1-7 provide the results of a two treatment protocol in accordancewith the present invention. According to this protocol, IL-12 wasdelivered by in vivo electroporation. C57BL/6 mice were treated withestablished subcutaneous B16.F10 melanoma by injecting 50 μg (1 μg/ml)of plasmid DNA encoding IL-12 (pIRES IL-12) in sterile saline into thetumor or the gastrocnemius muscle, followed by electroporation. Anapplicator containing six penetrating electrodes was used to deliver1500-V/cm, 100-μs pulses intratumorly. For intramuscular delivery, anapplicator, specifically designed for the mouse gastrocnemius muscle andcontaining four penetrating electrodes, was used to administer 100-V/cm,20-ms pulses, a protocol shown to result in high systemic IL-12 andIFN-γ expression. A single treatment did not result in long-term animalsurvival. Therefore, the following experiments administered a secondtreatment 7 days (day 7) after the initial treatment (day 0). Tumor sizewas evaluated throughout the experiment, and the results are presentedas the fold increase over day 0 tumor volume for each treatment group asshown in FIG. 1A. Treatment with pIRES IL-12 injected intratumor,followed by electroporation slowed tumor growth, with nearly half, 8 outof 17, of the mice showing complete regression of their tumors.Progressive tumor growth was observed in mice receiving intramuscularinjections of plasmid encoding IL-12 followed by electroporation. Micenot receiving electrical pulses, (P+E−), showed continued tumor growthuntil all mice were killed or succumbed to the tumor burden. Neither theadministration of electroporation alone (P−E+) nor intratumor (i.t.)delivery of a control vector (pND2Lux) with electroporation (V+E+)decreased tumor growth. These results provide evidence that neitherelectrical pulses alone nor plasmid DNA is responsible for tumorregression. None of the treatment groups except the P+E+ i.t. groupshowed tumor regression, although P+E− i.t. did show slower tumor growththan P−E− through day 14 (P<0.05).

Evaluation of mice 100 days after the initial treatment showed that 47%of mice, 8 out of 17, receiving intratumor delivery of IL-12 withelectroporation were tumor-free as shown in FIG. 1B. These mice wereconsidered cured. All mice receiving i.t. treatment with IL-12 andelectroporation experienced prolonged survival compared with animals inother treatment groups. None of the mice in the control groups survivedlonger than 35 days. Specifically, if left untreated or treated withpulses alone, mice did not survive longer than 21 days.

We challenged seven of the animals that showed complete regression andremained disease-free for 50 days in the right flank with B16.F10 tumorcells. No additional treatments were administered. Of the sevenchallenged, five were resistant to tumor growth on the right flank,while tumors grew in 100% of naive mice. This result suggests thedevelopment of an immune memory response following treatment of theinitial subcutaneous tumor established on the left flank.

As mentioned earlier, IL-12 induces several effects on the immunesystem. To evaluate the cytokine expression induced by eitherintramuscular or intratumor treatment, serum was analyzed and tumorlevels of IL-12 and IFN-γ. Serum levels of both cytokines were highestafter intramuscular injection followed by electroporation as illustratedby FIG. 2A. Serum IL-12 peaked at 320 pg/ml 10 days after treatment,whereas serum IFN-γ induced by IL-12 expression peaked at 177 pg/ml onday 14. Serum levels of both cytokines were significantly greater frommice treated intramuscularly with electroporation than other treatmentson days 5, 10, and 14 (P<0.05). Serum levels of these cytokines in micetreated with intratumor injection followed by electroporation were notsignificantly greater than expression in mice that received no treatment(P>0.05).

Analysis of IL-12 and IFN-γ expression within the tumors revealed thatintratumor treatment with electroporation resulted in the presence ofthese cytokines at the tumor site (FIG. 2B). Intratumoral IL-12 reached3 pg/mg of tumor tissue on day 5 and remained at that level through day10, whereas IFN-γ levels peaked at 8.16 pg/mg of tumor on day 5.Treatment with pIRES IL-12 injected intratumorly followed byelectroporation produced significantly higher (P<0.05) IFN-γ levels thanother treatment groups on days 5 and 10. Although tumor expression ofIL-12 reached 3 pg/mg of tumor with intratumor treatment, as opposed to0.64 pg/mg of tumor with intramuscular treatment, these levels were notsignificantly greater (P>0.05) as a result of a wide spectrum ofexpression levels in these tumors after intratumor treatment (0.5-6.9pg/mg of tumor tissue).

Treatment with intramuscular injection followed by electroporation didnot result in significant (P>0.05) cytokine expression within the tumorsas shown in FIG. 2B. Following intramuscular treatment the highest IFN-γexpression measured was 1 pg/mg of tumor on day 17. Therefore, treatmentprotocols that did not result in tumor regression also did not produceintratumoral IL-12 or IFN-γ expression. These results support previousreports on the critical need for cytokine expression within the tumor.

Resistance to challenge following successful tumor regression suggeststhe development of an immune memory response. The tumors were examinedhistologically 5 days after initial treatment to evaluate the influx ofimmune cells to the tumor. Tumor sections were stained with hematoxylinand eosin (H&E) to distinguish infiltrating immune cells from tumorcells. The H&E-stained sections showed infiltration of lymphocytes intothe tumors of mice 5 days after receiving intratumor injection of pIRESIL-12 followed by electroporation as shown in FIG. 3C.

In contrast, mice not treated or receiving intramuscular treatment withelectroporation did not display a great influx of lymphocytes asillustrated in FIGS. 3A and 3B. Treatment protocols not including invivo electroporation (P+E− either intratumor or intramuscular) also didnot result in the influx of lymphocytes (data not shown).

By immunohistochemical phenotyping, it is demonstrated that thelymphocytes observed in tumors following intratumor treatment with IL-12and electroporation were CD4+ and CD8+ T cells as illustrated in FIGS.4C and 4D. In comparison, lymphocytes were observed in limited numbersin untreated tumors as shown in FIGS. 4A and 4B. Treatment of mice withintramuscular injection followed by electroporation also resulted inlimited lymphocytic infiltrate, similar to that characterizing theuntreated control group of FIGS. 4E and 4F. Additionally, mice receivinginjection of plasmid encoding IL-12 (P+E− intratumor or intramuscular)or control plasmid with electroporation (V+E+ intratumor) did not showinfiltrating lymphocytes (data not shown).

To further evaluate the need for T lymphocytes in tumor regression,athymic nude mice deficient in T cells were used as the mouse model inplace of C57BL/6 mice. These mice were injected with B16.F10 tumor cellssubcutaneously and began treatment when tumors reached 3-5 mm indiameter. Mice received intratumor treatments as explained earlier:intratumor injections of plasmid encoding IL-12 without electroporation,intratumor injection of a control plasmid followed by electroporation,or intratumor injections of plasmid encoding IL-12 followed byelectroporation. Because of the lack of successful response in C57BL/6mice following intramuscular injection, we administered only intratumortreatments. None of the treatments in the nude mouse model resulted intumor regression as shown in FIG. 5A. In addition, no mice in anytreatment group survived longer than 30 days. This observation furthersuggests the necessity of a T-cell response for successful regression ofB16.F10 melanoma tumors.

Another potential role of IL-12 on tumor regression is its effect onangiogenesis. To assess the antiangiogenic role of IL-12 on B16.F10tumors in C57BL/6 mice, representative sections of three tumors fromeach treatment group were stained with anti-CD31 antibodies, markingendothelial cells. Five different areas of highest vascularity wereexamined at a magnification of ×400 for each group as illustrated byFIG. 6. A representative section of the vessels in an untreated tumor onday 0 is shown in FIG. 6A. FIGS. 6B and 6C show the large number ofvessels present within untreated tumors or tumors from mice receivingintramuscular injection followed by electroporation on day 5. Incontrast, FIG. 6D shows the reduction of blood vessels after intratumorinjection and electroporation on day 5. Tumors from mice receivinginjection of plasmid encoding IL-12 without electroporation (P+E−intratumor or intramuscular) or control plasmid with electroporation(V+E+) did not show a reduction in vasculature (data not shown).

In addition, vessels in each of the three tumors excised from untreatedmice were counted, mice receiving intramuscular IL-12 andelectroporation, and mice receiving intratumor IL-12 andelectroporation. In FIG. 7, Table 1 shows the number of blood vesselscounted in the field of highest vascularity at a magnification of ×400for each of the three excised tumors. Only intratumor injection followedby electroporation (P+E+ intratumor) resulted in significant (P<0.05)vessel reduction compared with untreated animals. Although anantiangiogenic effect was observed following intratumor treatment withelectroporation, the lack of response in the nude mouse model suggeststhat T cells may be a critical factor for obtaining regression ofB16.F10 melanoma. An antiangiogenic response may, however, contribute tostabilization of tumor size while an immune response is mounted.

This report has demonstrated that IL-12 delivered in the form of plasmidDNA with the aid of electroporation can result in successful regressionof B16.F10 tumors. The animals remain disease-free and are resistant tochallenge at a distant site. The results of the two treatment protocoldemonstrate nearly a 47% survival rate following gene therapy treatmentof established subcutaneous B16.F10 melanoma.

In summary, the present invention provide a treatment modality that caneradicate established B16.F10 melanoma tumors and result in resistanceto renewed tumor growth following challenge. Utilizing the two treatmentprotocol, after i.t. delivery of plasmid DNA encoding IL-12 by in vivoelectroporation, 47% of mice showed complete regression of their tumorsand remained disease-free. These mice were challenged with B16.F10 tumorcells, and five of seven remained tumor-free for an additional 100 days,after which they were humanely killed. Also, it is demonstrated thati.t. injection of plasmid DNA encoding IL-12 and electroporation is moreeffective than i.m. delivery for promoting tumor regression andprolonging animal survival. The success of this treatment in this tumormodel stems from the local expression of IL-12 and IFN-γ, infiltratinglymphocytes, and inhibition of angiogenesis within the treated tumor.

FIGS. 8-12 are illustrative of the three-treatment protocol inaccordance with the present invention. Regarding the short-termprevention of subcutaneous tumors at a distant site, C57B1/6 mice wereshaved on both flanks Mice were injected subcutaneously in the leftflank with 1×10⁶ B16.F10 cells in 50 μl of sterile PBS. Once tumors wereestablished, measuring 3-5 mm in diameter, treatment was begin. Twotypes of experiments were performed. The first series of experiments, onthe day of the first treatment, 5×10⁵ B16.F10 cells in 50 μl of sterilePBS were injected in the right flank of mice. The second set ofexperiments, 5×10⁵ B16.F10 cells in 50 μl of sterile PBS were injectedin the right flank of mice three days after the left flank injection.For both sets of experiments, mice received intratumor or a combinationof intratumor and intramuscular therapy to the initial tumor on the leftflank as described previously. Pulse protocols are further describedwithin the results section. Established tumors on the left flank werecontinuously measured as described earlier, and the right flanks of themice were monitored for tumor development.

Regarding the analysis of lung colonization, B16.F10 cells were preparedas previously detailed for subcutaneous injection. Either 1×10⁵ or 5×10⁵B16.F10 cells in 50 μl of sterile PBS were injected into the tail veinusing a 1 cc syringe with a 30-gauge needle. Mice receivedintra-muscular treatment on the day of inoculation and four days lateras described earlier. Twenty-one days following inoculation, mice wereeuthanized and their chest cavities exposed. Lung colonies appeared asblack tumor nodules on the lung surface and were counted.

As shown previously with the two-treatment protocol, a 47% disease-freesurvival rate for greater than 100 days in mice bearing establishedsubcutaneous B16.F10 tumors treated twice with i.t. injection of plasmidencoding IL-12 and electroporation. Five out of seven disease free micewere resistant to challenge following an additional inoculation of tumorcells in the opposite flank. We previously noted a poor response to thefirst treatment was often observed in tumors that did not fully regress.By the second treatment seven days later, these tumors had shownextensive growth and could possibly have been too large for successfulregression by the additional treatment. Increases in the disease freesurvival rate were obtained by two methods. First, instead of twotreatments three treatments were delivered to these mice on days 0, 4,and 7. Second, an intra-muscular treatment was added. As discussedearlier, it has been shown that intra-muscular delivery of IL-12 plasmidresults in a systemic production of IL-12 and IFN-γ (41). These micealso received three treatments.

The administration of three-treatments, whether i.t. alone or incombination with i.m., resulted in complete tumor regression and anincreased disease free survival rate over two treatments as illustratedby FIG. 8. Both three-treatment protocols (i.t. alone or i.t. and i.m.)resulted in an 80% disease free survival rate, statistically significant(p<0.05) over the 60% disease free survival rate resulting from thetwo-treatment protocol (FIG. 1b ). The slight increase in the diseasefree survival rate with the two-treatment protocol over our previousresults with two treatments (60% vs. 47%) was not statisticallysignificant. All three of the treatment protocols delivering IL-12plasmid by electroporation resulted in complete regression of the tumorsand maintenance of a disease free status through 100 days. Whenchallenged with B16.F10 cells, all 12 (100%) of the disease free mice inthe three treatment groups were resistant, and eight out of nine mice(88.9%) in the two-treatment group were resistant, suggesting thedevelopment of an immune memory response. These treatment protocols werefurther examined in multiple tumor and metastatic models.

The experiments described above demonstrated that the formation of newtumors (opposite flank) could be prevented in a high percentage of micethat had a complete response and long-term disease free survival. Tofurther examine the potential of this therapeutic approach, it wasimportant to evaluate the ability to block the formation of new tumorsprior to the regression of the primary tumor. On the same day that micereceived the first treatment for an established B16.F10 tumor on theleft flank, a second injection of B16.F10 cells were administered to theright flank. Mice were then evaluated for regression of the first tumoras well as prevention of establishment of the second tumor.

Treatment protocols that involved i.t. or i.t./i.m. injections andelectroporation resulted in regression of the primary tumors as well asprevention of the establishment of the secondary tumor (FIG. 9a, b ).Secondary tumors developed in 27% mice receiving two treatments and 33%mice receiving either of the three treatment protocols (FIG. 9a ). Ofthe mice receiving an i.t. injection of control plasmid followed byelectroporation, 77% of mice developed secondary tumors. In the notreatment group, 55% mice developed the secondary tumor and 58% micegrew the second tumor in the group receiving i.t. injection only (FIG.9a ). Because of the aggressiveness of this tumor model, several mice inthe control treatment groups succumbed to their primary tumor before thesecondary tumor could develop. Therefore, the percentage of micedeveloping the secondary tumor may have been higher in these groups hadthe mice survived for a longer period of time. Survival (FIG. 9b ) wassignificantly improved (p<0.01) in all three groups that received bothIL-12 plasmid and electroporation compared to no treatment, plasmidinjection alone and injection of control plasmid followed byelectroporation. The mean survival for each group was as follows: notreatment=17.9+/−6.7 days; plasmid injection alone=30.1+/−28.9 days;control plasmid followed by electroporation=20.6+/−6.0 days; i.t. andi.m. plasmid injection and electroporation (3 treatments)=59.5+/−27.7days; i.t. plasmid injection and electroporation (2treatments)=65.2+/−24.0 days; i.t. plasmid injection and electroporation(3 treatments)=68.6+/−31.8 days. Seven out of 15 (47%) mice treated withi.t. plasmid injection and electroporation (3 treatments) wereconsidered “cured” as they had no evidence of disease 100 days posttreatment. In the i.t./i.m. three treatment and the i.t. two treatmentgroup 4 out of 15 (26%) were “cured”.

A second series of experiments was performed to examine if this approachcould prevent formation of distant subcutaneous tumors when the tumorcells were injected prior to treatment. Three days after mice receivedan injection of B16 cells in the left flank (approximately four daysbefore mice received treatment for the established B16.F10 tumor on theleft flank) we administered a second injection of B16.F10 cells to theright flank. As in the previous experiment, mice were evaluated forregression of the first tumor as well as prevention of establishment ofthe second tumor (FIGS. 10 a, b). Secondary tumors developed in 50% ofmice receiving two or three i.t. treatments and 25% of mice receivingthree i.t. and i.m. treatments (FIG. 10a ). In the control groups: 100%of mice receiving i.t. injection of IL-12 plasmid withoutelectroporation, 87.5% of the no treatment group and 75% of the micereceiving an i.t. injection of control plasmid followed byelectroporation developed secondary tumors (FIG. 10a ). A significantincrease (p<0.05) in survival was seen only in mice receiving three i.t.or i.t./i.m. treatments (FIG. 10b ) compared to the 3 control groups.Survival of mice in the i.t. two treatment group was not significantlydifferent than any of the other groups. The mean survival for each groupwas as follows: no treatment=21.8+/−4.8 days; plasmid injectionalone=26.0+/−8.8 days; control plasmid followed byelectroporation=23.5+/−6.6 days; i.t. and i.m. plasmid injection andelectroporation (3 treatments)=37.9+/−11.2 days; i.t. plasmid injectionand electroporation (2 treatments)=38.1+/−24.5 days; and i.t. plasmidinjection and electroporation (3 treatments)=47.8+/−26.1 days. Only twomice, one in the i.t./i.m. group and the other in the i.t. two treatmentgroup were tumor free at 100 days and considered “cured”.

B16.F10 melanoma cells will form tumor nodules in the lungs after i.v.injection. Treatment of this model requires a protocol that does notinvolve a primary or subcutaneous tumor. Therefore, the proposed therapymust induce a systemic immune response that can respond to the tumorburden in the lungs. We showed previously that i.m. injection of IL-12plasmid followed by electroporation results in high serum levels ofIL-12 and IFN-γ. Furthermore, these serum levels could be sustained fora longer period by adding a second treatment four days after the initialtreatment.

In this model, C57B1/6 mice i.v. with 1×10⁵ B16.F10 cells was injectedand administered i.m. treatment with 50 μg of plasmid encoding IL-12 andelectroporation. Four days following the injection and initialtreatment, we administered a second treatment. Mice were euthanized 21days later and their lungs examined for tumor nodules. The table of FIG.11 shows that 37.5% of mice receiving treatment with IL-12 andelectroporation developed lung colonies. Of those three, two micepresented with only one nodule. In contrast, 87.5% of mice not treateddeveloped lung colonies and 75% of mice receiving i.m. injection ofplasmid encoding IL-12 without electroporation or mice receiving i.m.injection of a control plasmid with electroporation developed tumornodules.

To evaluate the efficacy of this treatment on a heavier tumorinoculation, 5×10⁵ B16.F10 cells were injected, i.v. then administeredtreatments as described above. Because the mice in control groups begandying before 21 days, the data is shown as survival (FIG. 11). 100% ofmice in the group receiving i.m. injection of plasmid encoding IL-12with electroporation survived throughout the experiment. Of the controlgroups, 62.5% in the no treatment group survived, 75% mice in theinjection only group survived, and 50% mice in the group receivingcontrol plasmid followed by electroporation survived. Thus, i.m.injection of plasmid coding for IL-12 followed by electroporationresults in the establishment of fewer lung colonies and increasessurvival of mice with a heavy tumor inoculation.

With reference to FIG. 13, the results of the treatment of melanomatumors in a mouse model using plasmid coding for B7-1 followed byelectroporation of 1300 V/cm and 100 μs pulses. Similar protocols aspreviously presented using plasmid coding for IL-12 and IL-15 may beused with plasmid coding for B7-1.

In accordance with the present invention is demonstrated delivery ofplasmid encoding IL-12 and B7-1 by electroporation results in successfultreatment of subcutaneous tumors as well as lung metastases. We havealso shown that this approach is not only effective in treatingestablished tumors but is also effective in preventing the formation ofnew tumors. The results also suggest that this approach may be useful intreating multiple subcutaneous tumors. There was a reduction in theformation of distant second tumors when only the primary tumor wastreated. This effect was seen when the tumor cell injection occurred onthe same day of treatment or 4 days prior to treatment. Althoughadministration of other electroporation protocols, using plasmid IL-12and B7-1, have shown some regression or delay of tumor growth, thetreatment protocols presented here have shown the highest rate ofsuccess against murine B16.F10 melanoma.

The lack of adverse side effects from the administration of theelectrical pulses themselves is an enticing factor for its use. Phase Iand II human clinical trials administering electrical pulses for thedelivery of chemotherapeutic agents showed success against local tumors.General anesthesia was not required, and the patients did not report anyserious adverse events. During the administration of the pulses,patients acknowledged feeling individual pulses but did not report anyresidual sensation. Thus, the use of electrical pulses is certainlyapplicable to human use.

Furthermore, for gene therapy studies, electroporation can effectivelyenhance the delivery of naked DNA. Plasmid DNA does not require celldivision, nor has it elicited serious toxicities or immune responsescompared to delivery of recombinant protein or the use of viral vectors.As mentioned previously, Lohr et al. compared delivery of IL-12 byelectroporation to adenoviruses and found significantly less sideeffects in the mice following treatment protocols with electroporation.While the use of in vivo electroporation for delivery of plasmid DNA isin a relatively early stage of development, there have been severalpre-clinical studies that suggest this approach may be useful againstseveral cancer types. The present invention provides a method for theadministration of a plasmid encoding IL-12 and B7-1 with electroporationhas a therapeutic effect on primary tumors as well as distant tumors andmetastases.

Material and Methods for IL-15

Mice, cell lines and plasmids. The human IL-15 expression plasmid(pIL-15) used was optimized for maximal expression and was 80-fold moreefficient than standard pcDNA3-based plasmids. In addition, this humanIL-15-expressing plasmid was demonstrated to be approximately 70%homologous to murine IL-15 and was shown to enhance antigen-specificCD8+ immune responses in mice. Also, it has been shown that the humanIL-15, generated from the plasmid, did not induce murine anti-humanIL-15 antibodies after injection into mice.

In accordance with the present invention, the strategy for plasmidoptimization involved the insertion and replacement of the existingKozak sequence with a stronger Kozak sequence as well as removingupstream inhibitory AUGs through primer design. In addition to thesechanges, the native long signal peptide sequence was replaced by anoptimized leader sequence, which had been shown to enhance secretion andexpression of the protein. Subsequently, the optimized IL-15 plasmid wasinserted into a cloning vector, which contains a ubiquitous andconstitutively active promoter. In the experiments reported here, theoptimized IL-15 plasmid has been designated pIL-15. The DNA generatedfor use in these experiments was produced using endotoxin-free ClontechGiga (Clontech, Palo Alto, Calif.) kits.

In the present invention, C57BL/6 mice, the murine strain syngeneic forthe B16F10 melanoma tumor cell line, were used and were purchased fromthe National Cancer Institute. Mice were housed and maintained duringthis study in accordance with Association for Assessment andAccreditation of Laboratory Animal Care (AAALAC) guidelines. The B16.F10murine melanoma cell line clone (CRL 6475) was originally purchased fromATCC and was maintained for studies as monolayers in culture in 90%McCoy's medium supplemented with 10% fetal bovine serum. For thepreparation of the single-cell suspension for tumor induction,monolayers of cells were detached from flasks usingtrypsin-ethylenediaminetetraacetic acid.

Tumor Induction and Measurement.

Tumors were induced by the subcutaneous injection of 10⁶ B16.F10 cells(greater than 90% viability by Trypan blue exclusion) into the leftflanks of C57BL/6 mice. Tumors were permitted to grow to an average size(i.e. volume) of 40 mm³ before initiation of the treatment regimen. Thisapproximate tumor volume has been determined to be an ideal minimal sizefor intratumoral injection as the administered treatment volume isretained effectively within the lesion with no significant leakage,providing confidence that the entire dose had been administered. Thismean tumor volume for initial therapeutic intratumoral injection hasbeen used in previous studies. Tumor volumes were determined before andat periodic intervals following treatment, using a digital caliper bymeasuring the longest diameter (a) and the next longest diameter (b)perpendicular to (a). Using these measurements, the tumor volume wascalculated by the formula: V=ab²×π/6. The mice were followed in theexperiments for 100 days or until tumor volume was determined to be 1300mm³ at which point any mice had usually succumbed to tumor burden orwere requisitely and appropriately euthanized owing to the size of thetumor.

Intratumoral Plasmid Treatment and In Vivo Electroporation.

Female C57BL/6 mice, 6-7 weeks old were injected with the B16.F10melanoma cells as indicated above and tumors were allowed to grow to therequired size. Tumors were then treated intratumorally with either 50 μgof the pIL-15 or the backbone plasmid vector. Subsequently (i.e. within1 min), tumors from the appropriate groups were subjected to in vivoelectroporation using a custom-made applicator, containing sixpenetrating electrodes that was inserted into the tissue around thetumor and six pulses that were 100 μs long at a field strength of 1500V/cm were administered using a BTX T820 pulse generator (BTX HarvardApparatus, Hollister, Mass.) and autoswitcher (Genetronics, San Diego,Calif.).

As indicated, treatments were administered on days 0 and 4 with pIL-15at a dose of 50 μg. For the treatment, groups P+ or P− indicates with orwithout treatment with the pIL-15 plasmid and E+ or E− indicates with orwithout electroporation, respectively. V+ designates the control‘backbone’ vector, at a dose of 50 μg, which was delivered withelectroporation. The treatment groups were as follows: P−V−E−=notreatment, P−V+E+, P+V−E− and P+V−E+. The results presented are the meanresults of a total of 16 mice for each group from two separateexperiments.

The mean tumor volumes were calculated for each group at selected timepoints after the treatment regimen up to day 100 after the initiation ofthe treatment regimen. Additional quantitative measurements made werefold increase in tumor volume compared to day 0 as well as percent ofmice undergoing complete tumor regression coupled with long-termsurvival.

Expression of Intratumoral IL-15 after Treatment with pIL-15.

In order to access the effect of in vivo electroporation on intratumoralexpression of IL-15 after delivery of pIL-15, an enzyme-linkedimmunosorbent assay (ELISA) assay was utilized. Briefly, three groups offour C57BL6 mice each were injected with 10⁶ B16.F10 cells as describedabove. The tumors were allowed to develop to the appropriate size (i.e.40 mm3). One group was untreated, whereas the second and third groupswere treated with 50 μg of pIL-15 with or without concomitant in vivoelectroporation respectively. Thirty-six hours later, animals werekilled and tumors were removed and homogenized by sonication inphosphate-buffered saline containing a protease inhibitor cocktail. Therationale for the 36 hour time point was based upon in vitro expressionstudies with pIL-15 in other tumor cell lines, which indicated thatexpression of IL-15 peaked at 36-48 hours. IL-15 levels were thenmeasured in the tumor homogenates/lysates with a human Duo IL-15 ELISAkit (R and D Systems Inc., Minneapolis, Minn.) and expressed as specificpg of IL-15/mg tumor. Data indicated are the mean of fourquadruplicates. In addition, sera samples were collected from treatedmice and assayed for IL-15 expressed from pIL-15.

Histological Analysis of Sections from pIL-15-Treated Tumors.

An additional study was performed, which examined tumor sections frommice treated with pIL-15. In this study, four groups of mice (n=6) thatdiffered in the treatment regimens (P−V−E−, P+V−E−, P−V+E+ and P+V−E+)were treated on days 0, 4 and 7. Forty-eight hours after the finaltreatment, the mice were killed after which the tumors were excised,fixed in 10% formalin and sectioned. The sections were stained forhistological analysis with hematoxylin and eosin by standard methods andexamined microscopically for the presence of tumor cells, necrosis aswell as lymphocytic infiltration.

Statistical Analysis.

Among the different treatment groups, the mean tumor volume was used inthe calculation of mean fold increase in tumor volume for the selectedtime-point assessment as compared to day 0. Statistical analysis of anytreatment differences, as measured by mean fold tumor volume increase,was made using Student's t-test methods.

The initial experiment reported here addressed the hypothesis thatintratumoral electroporation of subcutaneous B16 melanoma tumors willenhance the expression of IL-15 from an IL-15 DNA expression plasmid. Asindicated above, 50 μg of the pIL-15 was injected intratumorally intosubcutaneous B16.F10 melanoma tumors of the appropriate tumor volume ineither the absence (P+V−E−) or presence (P+V−E+) of subsequentintratumoral electroporation. Thirty-six hours later, tumors wereexcised and homogenized and IL-15 concentrations were measured andexpressed as pg IL-15/mg tumor. The background IL-15 concentration intumor homogenates of untreated (i.e. no pIL-15 or electroporation) was6.0 pg IL-15/mg tumor. FIG. 14 shows intratumoral IL-15 concentrationsof 9.4 and 28.1 pg/mg tumor in the P+V−E− and P+V−E+ groups,respectively. This result indicates that in vivo electroporationenhanced intratumoral expression of IL-15 approximately 4.7-fold,compared to non-treated controls, and threefold compared to pIL-15treatment without electroporation. In this invention, the levels ofIL-15 measured in both the P+V−E− and P+V−E+ groups were statisticallyelevated (P<0.05 by the Student's t-test) when compared to the levelmeasured in the P−V−E− group. Also, and importantly, the intratumoralIL-15 level measured in the P+V−E+ group was significantly elevatedcompared to that measured in the P+V−E− group. These results demonstratethe ability of the pIL-15 plasmid to be expressed within the tumor afterintratumoral delivery with additional significant enhancement ofexpression through delivery by in vivo electroporation. However, at this36 h post-treatment time point, IL-15 expressed from this plasmid wasnot measurable in sera samples from mice from the appropriate treatmentgroups (data not shown).

It was subsequently relevant to determine the potential therapeuticefficacy of intratumoral delivery of pIL-15 and whether in vivoelectroporation could enhance any antitumor therapeutic effect ofpIL-15. Therapeutic end points in these experiments are ‘slowing’ oftumor growth, as measured by tumor volume, as well as by the incidenceof complete regressions of tumors coupled with the long-term survival.In this experiment, C57BL/6 mice were injected subcutaneously withB16.F10 melanoma cells. When tumors had attained the appropriate volumemice were separated into four groups (n=16 each) and treated. Inuntreated controls, the tumors grew rapidly, which is characteristic ofthe B16.F10 clone, and all of the tumors reached a volume ofapproximately 1000 mm³ by day 18.

As shown in the table of FIG. 15, data for the other groups at day 18after the initiation of treatment are summarized. The day 18 measurementwas selected because at this time point at least 50% of the mice in eachof the treatment groups were still alive (i.e. had not succumbed totumor burden) allowing for a meaningful analysis. The untreated control(P−V−E−), as indicated above, had undergone an average 22-fold increase(i.e. from 46.5 to 1026.8 mm3) in tumor volume at day 18 compared to day0. By contrast, in the P+V−E+ group, the mean fold tumor volume increasefrom day 0 to day 18 was only 1.2 (i.e. from 39.9 to 49.6 mm3). Inaddition to these findings, there appeared to be some tumor growthslowing/attenuation effect in the other treatment groups as well at day18 compared to day 0. That is, the fold increase in tumor volume fromday 0 to day 18 in groups P−V+E−, P−V+E+ and P+V−E− was 12.1, 4.2 and15.6, respectively. These results indicate an initial ‘nonspecific’vector backbone and electroporation effect on tumor growth. However, thegrowth attenuation effect in the P+V−E+ group, receiving the IL-15expressing plasmid, was significantly greater in terms of fold increasein tumor volume, compared to any of the other treatment groups whenaccessed by a Student's t-test (P<0.05). The growth attenuation effectnoted with control vector treatment or electroporation alone has beennoted previously and has been attributed to effects of CpG motifs withinthe vector and an initial inflammatory/toxic effect of theelectroporation. It is relevant to point out, however, that at day 18the percent of mice in the P+V−E+ group, which had undergone tumorregression, was significantly higher (i.e. 62.5%) than any of the othertreatment groups.

Ultimately, the endpoint for this study with the most relevant clinicalsignificance is complete tumor regression coupled with long-termsurvival of the mice. Time-point measurements of percent mouse survivalwith complete tumor regression within the different treatment groupswere performed up to 100 days after the initiation of the experiment.For the B16.F10 murine melanoma tumor model, complete tumor regressionand animal survival 100 days post-tumor cell injection has beengenerally accepted as the benchmark for ‘curative’ therapeutic regimens.That is, maintenance of complete tumor regression 100 days after theinitiation of treatment can be considered to be a long-term ‘cure’.These data are summarized in the Kaplan-Meier survival curves shown inFIG. 16. The data summarized in the graph indicates that at the day 100time point only the P+V−E− and P+V−E+ treatment groups had survivinganimals with complete tumor regressions. Mice in all of the other groupshad succumbed to tumor burden by that time point. In the P+V−E− group,2/16 (12.5%) of the mice survived until at least the day 100measurement. In contrast, the P+V−E+ group had 6/16 (37.5%) of thetreated animals surviving to at least day 100. This was a threefoldenhancement of the therapeutic effect compared to pIL-15 treatmentwithout electroporation. This enhancement was statistically significantat the 0.05 level when measured by a Student's t-test. Importantly, inthe control groups that were treated with the vector backbone with orwithout electroporation, none of the animals survived long term withcomplete tumor regressions. In addition, no tumor-bearing mice thatreceived electroporation alone (data not shown) survived long term withcomplete tumor regression. These results demonstrated that the pIL-15treatment was able to mediate complete tumor regression/long-termsurvival in B16 melanoma bearing C57BL/6 mice. Also, the end pointtherapeutic effect of pIL-15 was also enhanced by electroporation,indicating the clinical potential of this delivery method.

As indicated in the Materials and Methods section, an additional studywas performed, which examined tumor sections from the various groupshistologically 48 h after the final treatment. This was carried out inorder to access for the presence of melanoma tumor cells. Results of thehistological analysis indicated that in the P−V−E− control group therewas evidence of tumor in all of the mice, whereas in the P+V−E+ group83% of the mice failed to demonstrate histologic evidence of melanoma.In the P−V+E+ and P+V−E− groups, only 17% of the mice in each groupfailed to demonstrate evidence of tumor. These data further establishthe therapeutic efficacy of treatment with pIL-15 and in vivoelectroporation.

An extension of the regression/long-term survival study reported herewas performed in which long-term survivors were challengedsubcutaneously with 10⁶ B16.F10 melanoma cells. This experiment wasconducted in order to determine whether mice cured of their initialtumors through treatment could resist re-challenge with the B16.F10 cellline. Resistance to re-challenge would likely assume that animmunological mechanism (e.g. T-cell immune response) was operant andwhich putatively could be involved in protection. In this study,surviving mice from the study described in FIG. 16 were ‘challenged’shortly after the day 100 time point with the melanoma cells and wereaccessed over time for the development of tumors. The resultsdemonstrated that 60% of mice re-challenged from the P+V−E+ groupremained tumor free for at least 50 days, whereas likewise one of thetwo mice (i.e. 50%) from the P+V−E− group failed to develop a tumor inthis time span. The results from this limited re-challenge study, whilenot statistically significant, can be considered to be biologicallysignificant because of the aggressive nature of the B16.F10 melanomacell line. That is, naive mice injected with the number of B16.F10melanoma cells used in these experiments would normally succumb to tumorburden within approximately 20 days. As such, these findings suggestedthat pIL-15 treatment mediated an immunological response that protecteda proportion of the surviving/cured mice from tumor re-challenge.

The present invention demonstrates the therapeutic antitumor potentialof the IL-15-expressing plasmid when delivered intratumorally intoestablished subcutaneous B16 melanoma tumors in C57BL/6 mice. Inaddition, it was also demonstrated that delivery of pIL-15 with in vivoelectroporation significantly enhanced the antitumor activity of thisexpression plasmid, which was associated with an approximate threefoldand 4.7-fold increase in expression of IL-15 when delivered withelectroporation as compared to treatment without electroporation or notreatment, respectively. Also, it appeared that the plasmid backbonevector, when delivered by electroporation resulted in an initialtemporary attenuation of tumor growth due likely owing to immunestimulatory effects of the CpG motifs contained in the plasmid as wellas an inflammatory response from the electroporation procedure. However,only treatment with pIL-15 resulted in any complete tumor regressionswith long-term survival, indicating specificity in mediating thisrelevant endpoint therapeutic response. It is anticipated that furtherstudies with electroporative delivery of pIL-15 will allow maximization(i.e. at least an 80% complete tumor regression/long-term survival rate)of the therapeutic response. Therapeutic maximization strategies includemodulation of the dose as well as the number and intervals oftreatments.

Re-challenge with B16 melanoma cells of mice that had been ‘cured’ ofthe initial melanoma tumors by treatment with pIL-15 pluselectroporation resulted in resistance to tumor challenge in a largeproportion of the mice. This suggested that a mechanism resulting inimmunological memory mediated the resistance to tumor cell challengeeven though preliminary analysis at the 36 h post-treatment time pointfailed to demonstrate sera levels of expressed IL-15. Future studies inthis area will be aimed at further examining the tumoral and sera IL-15expression levels after treatment with pIL-15 plus electroporation aswell as the measurement of antigen-specific memory T cells or NK cellactivity as possible immunological mechanisms for mediating theantitumor activity of pIL-15.

The tumor induction and challenge model utilizing the B16.F10 cell line,as reported in this invention disclosure, is particularly relevant forseveral reasons: (a) the B16.F10 melanoma is a highly invasive,metastatic and poorly immunogenic tumor, which is very difficult to‘cure’, (b) the treatment regimen used in this study was administered toestablished tumors rather than injected concomitantly with tumor cellsor before malignant lesions had visibly formed and (c) the ultimatetherapeutic end point was complete tumor regression and long-termsurvival rather than simply attenuation of tumor growth. This isrelevant as the majority of other studies with this tumor cell line haveeither administered treatments before the development of visible tumorsor accessed tumor growth attenuation as the therapeutic end point.

As such, in accordance with the present invention, the potential utilityof naked DNA plasmids expressing therapeutic cytokines such as IL-15 asanticancer therapeutics has been described. In addition, the enhancementof the delivery, expression and therapeutic index for these molecularreagents through intratumoral electroporation has been established.

It will be seen that the objects set forth above, and those madeapparent from the foregoing description, are efficiently attained andsince certain changes may be made in the above construction withoutdeparting from the scope of the invention, it is intended that allmatters contained in the foregoing description or shown in theaccompanying drawings shall be interpreted as illustrative and not in alimiting sense.

It is also to be understood that the following claims are intended tocover all of the generic and specific features of the invention hereindescribed, and all statements of the scope of the invention which, as amatter of language, might be said to fall therebetween. Now that theinvention has been described,

What is claimed is:
 1. A method of increasing proliferation of T-celllymphocytes to at least one cancerous tumor comprising: injecting the atleast one cancerous tumor with an effective dose of at least one plasmidcoding for B7-1; and administering electroporation therapy to the tumor,the electroporation therapy comprising the administration of at leastone pulse having a field strength of at least 700V/cm and a duration ofless than 1 millisecond resulting in increased proliferation of theT-cell lymphocytes to the at least one tumor.
 2. A method of reducingangiogenesis and increasing proliferation of T-cell lymphocytes in atleast one cancerous tumor comprising: injecting the at least onecancerous tumor with an effective dose of at least one plasmid codingfor IL-12; and administering electroporation therapy to the tumor, theelectroporation therapy comprising the administration of at least onepulse having a field strength of at least 700V/cm and a duration of lessthan 1 millisecond resulting in reduction of angiogenesis.
 3. The methodof claim 2, wherein angiogenesis is reduced by at least 50% as comparedto an amount of angiogenesis present in an untreated cancerous tumor. 4.The method of claim 2, wherein angiogenesis is reduced between about 62%to about 75% as compared to an amount of angiogenesis present in anuntreated cancerous tumor.
 5. A method of reducing the occurrence of asecondary tumor in a subject having at least one primary cancerous tumorcomprising: injecting the primary cancerous tumor with an effective doseof at least one plasmid coding for IL-12 or a combination of IL-12 andB7-1; and administering electroporation therapy to the primary canceroustumor, the electroporation therapy comprising the administration of atleast one pulse having a field strength of at least 700V/cm and aduration of less than 1 millisecond resulting in the reduction of theoccurrence of the secondary tumor wherein the secondary tumor isseparate from the primary tumor.
 6. The method of claim 5, furthercomprising injecting the subject having at least one cancerous tumorintramuscularly with an effective dose of at least one plasmid codingfor IL-12 or a combination of IL-12 and B7-1 prior to the administrationof electroporation therapy.
 7. The method of claim 5, wherein theoccurrence of the secondary tumor is reduced by about 75%.
 8. The methodof claim 5, wherein the occurrence of the secondary tumor is reduced byabout 50%.
 9. The method of claim 5, wherein injecting the canceroustumor with an effective dose of at least one plasmid coding for IL-12 ora combination of IL-12 and B7-1 and administering electroporationtherapy to the tumor further comprises: administering a first treatmentat time T1, wherein the first treatment comprises injecting thecancerous tumor with a first effective dose of at least one plasmidcoding for IL-12 or a combination of IL-12 and B7-1 and administering afirst electroporation therapy to the tumor at time T1, the firstelectroporation therapy comprising the administration of at least onepulse having a field strength of at least 700V/cm and a duration of lessthan 1 millisecond; and administering a second treatment at time T2,wherein time T2 is a time later than time T1, wherein the secondtreatment comprises injecting the cancerous tumor with a secondeffective dose of at least one plasmid coding for IL-12 or a combinationof IL-12 and B7-1 and administering a second electroporation therapy tothe tumor at time T2, the second electroporation therapy comprising theadministration of at least one pulse having a field strength of at least700V/cm and a duration of less than 1 millisecond.
 10. The method ofclaim 9, wherein the duration between time T1 and T2 is seven days. 11.The method of claim 9, wherein the duration between time T1 and T2 isfour days.
 12. The method of claim 5, further comprising: administeringa third treatment at time T3, wherein time T3 is a time later than timeT2, wherein the third treatment comprises injecting the cancerous tumorwith a third effective dose of at least one plasmid coding for IL-12 ora combination of IL-12 and B7-1 and administering a thirdelectroporation therapy to the tumor, the third electroporation therapycomprising the administration of at least one pulse having a fieldstrength of at least 700V/cm and a duration of less than 1 millisecond.13. The method of claim 12, wherein the duration between time T2 and T3is three days.
 14. A method of treating a cancerous tumor comprising:injecting the cancerous tumor with an effective dose of at least oneplasmid coding for a combination of IL-12 and B7-1; and administeringelectroporation therapy to the tumor, the electroporation therapycomprising the administration of at least one pulse having a fieldstrength of at least 700V/cm and a duration of less than 1 millisecondresulting in the regression of the cancerous tumor and prevention offormation of new tumors.
 15. A method of treating at least one melanomatumor in a patient comprising: administering a first treatment to thepatient comprising: injecting the at least one melanoma tumor with afirst effective dose of at least one plasmid coding for IL-12; andadministering a first electroporation therapy to the at least onemelanoma tumor, the electroporation therapy comprising theadministration of six pulses having a field strength of 1500 V/cm and aduration of 10 μs; administering a second treatment to the patient fourdays after the first treatment was administered comprising: injectingthe at least one melanoma tumor with a second effective dose of at leastone plasmid coding for IL-12; and administering a second electroporationtherapy to the at least one melanoma tumor, the electroporation therapycomprising the administration of six pulses having a field strength of1500 V/cm and a duration of 10 μs; and administering a third treatmentto the patient three days after the second treatment was administeredcomprising: injecting the at least one melanoma tumor with a thirdeffective dose of at least one plasmid coding for IL-12; andadministering a third electroporation therapy to the at least onemelanoma tumor, the electroporation therapy comprising theadministration of six pulses having a field strength of 1500 V/cm and aduration of 10 μs wherein the administration of the three treatmentsresults in regression of the at least one melanoma tumor and preventionof formation of new tumors.